Mature oocytes of amphibians are arrested in meiotic metaphase II due to the action of a multicomponent cytostatic factor (CSF) {Masui, Y., Biochem. Cell Biol., 70:920-945 (1992); Sagata, N., et al., Nature (London); 342:512-518 (1989) and Shibuya, E. K., et al., Development, 106:799-808 (1989)}. CSF is part of a Ras-GTP signaling pathway which acts through a MAP kinase (MAPK) cascade {Daar, I., et al., Science, 253:74-76 (1991); Fukuda, M., et al., J. Biol. Chem., 269:33097-33101 (1994) and Pomerance, M., et al., J. Biol. Chem., 267:16155-16160 (1992)}. The arrest is released upon fertilization with the completion of meiosis, and the early embryo undergoes a series of rapid cleavages. A major component of CSF, the Mos protein kinase, is synthesized in abundance during oocyte maturation and activates MAPK via phosphorylation of MAPK kinase (MAPKK) {Sagata, N., et al., Nature (London), 342:512-518 (1989); Sagata, N., et al., Nature, 335:519-525 (1988); Posada, J., et al., Mol. Cell. Bio., 13:2546-2553 (1993); Haccard, O., et al., Science, 262:1262-1265 (1993); Matsuda, S., et al., J. Biol. Chem., 268:3277-3281 (1993) and Kosako, H., et al., J. Biol. Chem., 269:28354-28358 (1994)}. Mos, which appears to be germ-cell specific, is rapidly degraded following fertilization and is not observed in cleaving embryos {Sagata, N., et al., Nature, 335:519-525 (1988); Yew, N., et al., Nature (London), 355:649-652 (1991); Watanabe, N., et al., Nature, 352:247-248 (1991) and Watanabe, N., et al., Nature (London), 342:505-511 (1989)}. Upon fertilization, MAPK and MAPKK are inactivated and remain inactive until the later stages of embryonic development {Haccard, O., et al., Science, 262:1262-1265 (1993); Kosako, H., et al., J. Biol. Chem., 269:28354-28358 (1994) and Ferrell, J. E., et al., Mol. Cell. Biol., 11:1965-1971 (1991)}.
Maturation promoting factor (MPF), which is required for progression through the cell cycle, is stabilized by CSF {Sagata, N., et al., Nature 335:519-525 (1988) and Pickham, K. M., et al., Mol. Cell. Biol., 12:3192-3203 (1992)}. MPF consists of the cell division control 2 kinase (cdc2) and cyclin. MPF activity is high during metaphase II arrest; following fertilization, cdc2 is inactivated by cyclin degradation and cyclin is regenerated during each division cycle.
Protease activated kinase I (PAK I) is a unique multipotential serine/threonine protein kinase which has been found in an inactive form in all animals and tissues examined to date {Tahara, S. M., et al., J. Biol. Chem., 256:11558-11564 (1981); Tuazon, P. T., et al., Eur. J. Biochem., 129:205-209 (1982); Tuazon, P. T., et al., J. Biol. Chem., 259:541-546 (1984); Rooney, R. D. et al., FASEB J., 6:A1852 (1992); and Rooney, R. D., et al., FASEB J., 6:A1852 (1992)}. Studies with 3T3-L1 cells also led to the identification of an endogenously active form of the PAK I holoenzyme {Rooney, R. D., et al., FASEB J, 6:A1852 (1992)}.
To determine whether PAK I has a role in the regulation of the cell cycle and functions as a cytostatic protein kinase, the inactive holoenzyme and the proteolytically activated enzyme have been injected into one blastomere of 2-cell frog embryos {Rooney, R. D., et al., FASEB J., 7:A1213 (1993)}. Activated PAK I inhibited cleavage of the injected cell, while the noninjected cell continued dividing through late cleavage. Injection of the inactive holoenzyme or heat-treated PAK I has no effect on cell cleavage. The data suggests PAK I is a cytostatic protein kinase, involved in mediating inhibition of the cell cycle.
PAK I can be activated by limited proteolysis with trypsin or chymotrypsin {Tahara, S. M., et al., J. Biol. Chem., 256:11558-11564 (1981)} or a Ca.sup.2+ -stimulated protease {Tahara, S. M., et al., Eur. J. Biochem., 126:395-399 (1982)}, hence the initial nomenclature. The inactive holoenzyme has been identified and partially purified from rabbit reticulocytes {Tahara, S. M., et al., J. Biol. Chem., 256:11558-11564 (1981)} and skeletal muscle {Tuazon, P. T., et al., Eur. J. Biochem., 129:205-209 (1982)}, chicken gizzard {Tuazon, P. T., et al., J. Biol. Chem., 259:541-546 (1984)}, and 3T3-L1 cells {Rooney, R. D., et al., FASEB J., 6:A1852 (1992)}. PAK I from reticulocytes uses only ATP as a phosphoryl donor, with a K.sub.m apparent of 0.6 mM {Tahara, S. M., et al., J. Biol. Chem., 256:11558-11564 (1981)}. The enzyme requires sulfhydryl reducing agents to maintain activity. The optimal Mg.sup.2+ concentration for mixed histone as substrate is 45 mM; with less complex substrates, the Mg.sup.2+ optimum is 5-10 mM {Tahara, S. M., et al., Eur. J. Biochem., 126:395-399 (1982)}.
Following activation in vitro by limited proteolytic digestion, PAK I phosphorylates histones H2B and H4 {Tahara, S. M., et al., J. Biol. Chem., 256:11558-11564 (1981)}, myosin light chain from skeletal and smooth muscle {Tuazon, P. T., et al., Eur. J. Biochem., 129:205-209 (1982) and Tuazon, P. T., et al., J. Biol. Chem., 259:541-546 (1984)}, translational initiation factors eIF-3, eIF-4B, and eIF-4F {Tahara, S. M., et al., Eur. J. Biochem., 126:395-399 (1982) and Tuazon, P. T., et al., J. Biol. Chem., 264:2773-2777 (1989)}, and avian and Rous sarcoma virus nuclear capsid protein NC {Leis, J., et al., J. Biol. Chem., 259:7726-7732 (1984); Fu, X., et al., J. Biol. Chem., 260:9941-9947 (1985) and Fu, X., et al., J. Biol. Chem., 263:2134-2139 (1988)}. In myosin light chain from smooth muscle, PAK I phosphorylates the same site as the Ca.sup.2+ /calmodulin-dependent myosin light chain kinase {Tuazon, P. T., et al., J. BioL Chem., 259:541-546 (1984)}. Phosphorylation of PAK I stimulates the actin-activated Mg-ATPase activity of myosin to the same extent as that observed upon phosphorylation of actomyosin by the Ca.sup.2+ -dependent myosin light chain kinase. An alternative site is modified in myosin light chain from skeletal muscle {Tuazon, P. T., et al., Eur. J. Biochem., 129:205-209 (1982)}.
In the Rous sarcoma virion, NC is fully phosphorylated at serine 40 {Fu, X., et al., J Biol. Chem., 260:9941-9947 (1985)}; following dephosphorylation, serine 40 is specifically modified by PAK I {Leis, J., et al., J. Biol. Chem., 259:7726-7732 (1984)}. Phosphorylation enhances the affinity of NC for single-strand RNA up to 100-fold by altering the conformation, allowing basic residues N-terminal to serine 40 to interact with RNA {Fu, X., et al., J. Biol. Chem., 260:9941-9947 (1985)}. Data with site-specific mutants of serine 40 indicate this residue is the switch between tight and loose binding {Fu, X., et al., J. Biol. Chem., 263:2134-2139 (1988)}.
It is an object of the present invention to provide compositions comprising PAK I or active fragments thereof and methods for the use of such compositions.